The aim of this proposal is to delineate antecedent cellular and molecular events that lead to the dysfunction of the endocytic, autophagic, and lysosomal systems (EALS), the earliest cellular disturbances known to occur in sporadic Alzheimer's disease (AD). The design is to assess gene expression levels within vulnerable populations while avoiding potential contamination from other cell types. Gene expression is assayed in neurons with early endosomal abnormalities, the first sign of AD-related responses, as compared to not-as-yet affected neighbors and to less vulnerable neuronal populations. A "molecular fingerprint" of human hippocampal neurons and neocortical neurons, mouse hippocampal and neocortical neurons, and fibroblasts is performed on human postmortem brains, a mouse model of Down's syndrome (Ts21) termed Ts65Dn, and in cultured cells. In this manner, cDNA array analysis is applied systematically to characterize gene expression changes at the inception of EALS pathology relative to spared neurons in these brains and to unaffected neurons in normal control brains. The experimental design entails microaspiration of identified neuronal populations followed by a novel single cell RNA amplification methodology developed in the laboratory of the Project Leader combined with custom-designed cDNA array analysis. Aim 1 consists of assessment of select neuronal populations from human postmortem brains. Aim 2 evaluates individual neuronal populations obtained from Ts65Dn and diploid mice. Aim 3 consists of gene expression analysis of cultured Ts21 fibroblasts with App levels knocked down via small interference RNA (siRNA). Aim 4 utilizes siRNA technology to knockdown other genes in the trisomic region for subsequent cDNA array analysis. This state-of-the-art paradigm enables an extensive, concurrent representation of hundreds of genes in selectively vulnerable and relatively spared cell types to neurodegeneration that represent some of the earliest pathological changes observed in AD and Ts21 brains at defined stages of pathology evolution.